Nuclear factor κB (NF-κB) is a family of inducible eukaryotic transcription factor complexes participating in regulation of immune response, cell growth, and survival [Ghosh et al. 1998]. The NF-κB factors are normally sequestered in the cytoplasmic compartment by physical association with a family of cytoplasmic ankyrin rich inhibitors termed IκB, including IκBα and related proteins [Baldwin et al. 1996]. In response to diverse stimuli, including cytokines, mitogens, and certain viral gene products, IκB is rapidly phosphorylated at serines 32 and 36, ubiquitinated and then degraded by the 26S proteasome, which allows the liberated NF-κB to translocate to the nucleus and participate in target gene transactivation [Mercurio et al 1999, Pahl et al 1999]. Recent molecular cloning studies have identified a multi subunit IκB kinase (IKK) that mediates the signal-induced phosphorylation of IκB. The IKK is composed of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit IKKγ. The catalytic activity of both IKKα and IKKβ can be activated by a multitude of different NF-κB inducers, including the inflammatory cytokines, tumor necrosis factor and interleukin-1, the T cell receptor and the T cell costimulatory protein, CD28 [Karin et al 2000].
NF-κB-inducing kinase, NIK, (MAP3K14) is a mitogen activated protein kinase (MAP3K) that was discovered by applicants in 1996 (WO9737016) while screening for proteins that bind to the TNF-receptor associated adaptor protein TRAF2 [Rothe et al. 1994, Takeuchi et al. 1996]. Marked activation of NF-κB upon overexpression of this protein kinase, and effective inhibition of NF-κB activation in response to a variety of inducing agents (LMP1, TNFR1, TNFR2, RANK, hTollR, CD3/CD28, interleukin-1R, human T-cell lymphotropic virus-1 Tax, LPS and others [Mlinin et al. 1997, Sylla et al 1998, Darnay et al. 1999, Lin et al. 1999, Geleziunas et al. 1998] upon expression of catalytically inactive NIK mutants suggested that NIK participates in signaling for NF-κB activation [Mlinin et al. 1997].
Targeted disruption of the NIK gene [Yin et al 2001] and study of a naturally occurring mice strain with a point missense mutation in NIK (glycine to arginine at mNIK codon 855) [Shinkaura et al. 1999] revealed an essential role of NIK in lymphoid organ development, thus the mice mutant strain has been called ‘alymphoplasia (aly)’ mice. Both the aly/aly and NIK knockout mice manifest systemic absence of lymph nodes and Peyer's patches, disorganized splenic and thymic architectures, and immunodeficiency whose most resilient features are low serum Ig levels and lack of graft rejection [Shinkaura et al. 1999]. These abnormalities apparently reflect aberrant signaling by a variety of receptors. The developmental deficiencies of the NIK mutant mice resemble those found in mice deficient in the LTβ receptor (LTβR) suggesting that NIK participates in signaling by this particular receptor. Impaired B cell proliferative capacity in the aly/aly mice could be shown to correlate to a deficient response of these cells to LPS and CD40L [Garceau et al. 2000], and presence of excessive amounts of B1 cells in the mice peritoneal cavity could be ascribed to defects in homing of peritoneal cells to the gut associated lymphatic tissue system as a consequence of deficient chemokine receptor signaling in the secondary lymphoid tissue [Fagarasan et al. 2000].
Apart from these and probably other contributions to the regulation of the development and function of the immune system, NIK seems also to be involved in the regulation of various non-immune functions. The aly/aly (though not the NIK knockout) mice display deficient mammary gland development [Miyawaki 1994]. Moreover, in vitro studies implicated NIK in signaling that leads to skeletal muscle cell differentiation [Canicio et al. 2001] and in the survival and differentiation of neurons [Foher et al 2000].
Consistent with the suggested role of NIK as mediator of NF-κB activation, fibroblasts derived from aly/aly and NIK−/− mice fail to activate NF-κB in response to LTβR activation. Moreover, LTβR upregulation of VCAM-1, which occurs through NF-κB activation, is abnormal in aly/aly murine embryonic fibroblasts [Matsumoto et al. 1999]. Deficient phosphorylation of IκB has also been noted in the response of aly/aly B-lymphocytes to CD40 ligation. In contrast, in dendritic cells of these mice CD40-induced phosphorylation of IκB appeared normal [Garceau et al 1998]. Aly/aly peritoneal cells are also incapable of responding to the chemokine SLC with increased NF-κB activity [Fagarasan et al. 2000]. However, in none of the cells examined so far was the effect of TNF or IL-1 on NF-κB activation found to be ablated by NIK mutation.
Assessment of the pattern of the NF-κB species in lymphoid organs of aly/aly mice indicated that, apart from its role in the regulation of NF-κB complex(s) comprised of Rel proteins (A+p50) and IκB, NIK also participates in controlling the expression/activation of other NF-κB species. Most notably, the lymphocytes of the aly/aly mice were deficient of p52, an NF-κB species that is specifically formed in mature B-lymphocytes through proteolytic processing of an inactive precursor, p100 (NF-κB2), suggesting a deficiency in p100-p52 conversion [Yamada et al. 2000]. Indeed, NIK has been shown to participate in site specific phosphorylation of p100, both directly and through phosphorylation of IKKα, which in turn phosphorylates p100. This phosphorylation serves as a molecular trigger for ubiquitination and active processing of p100 to form p52. This p100 processing activity was found to be ablated by the aly mutation [Xiao et al. 2001, Senftleben et al. 2001].
In view of the structural homology of NIK to MAP3Ks, some attempts have been made to explore the involvement of NIK in the three other main protein kinase cascades known to involve MAP3Ks (the MAP kinase cascades: the ERK, JNK and p38 cascades) [Akiba et al. 1998]. Though in certain cells NIK seems not to participate in any of these cascades, some others cells (PC12) do appear to involve NIK in the ERK cascade [Fochr et al. 2000]. Evidence has also been presented that in certain cells NIK may participate in signaling to the phosphorylation of Jun, the downstream target of the JNK cascade, in a way that is independent of this particular cascade [Akiba et al. 1998, Natoli et al. 1997]. In all, these findings indicate that NIK indeed serves as a mediator of NF-κB activation, but may also serve other functions, and that it exerts these functions in a cell- and receptor-specific manner.
Like other MAP3Ks, NIK can be activated as a consequence of phosphorylation of the ‘activation loop’ within the NIK molecule. Indeed, mutation of a phosphorylation-site within this loop (Thr-559) prevents activation of NF-κB upon NIK overexpression [Lin et al. 1999]. In addition, the activity of NIK seems to be regulated through the ability of the regions upstream and downstream of its kinase motif to bind to each other. The C-terminal region of NIK downstream of its kinase moiety has been shown to be capable of binding directly to IKKα [Regnier et al. 1997] as well as to p100 [Xiao et al. 2001] and to TRAF2 [Malinin et al. 1997]. These interactions are apparently required for NIK function in NF-κB signaling. The N-terminal region of NIK contains a negative-regulatory domain (NRD), which is composed of a basic motif (BR) and a proline-rich repeat motif (PRR) [Xiao et al. 2000]. Apparently, the N-terminal NRD interacts with the C-terminal region of NIK in cis, thereby inhibiting the binding of NIK to its substrate (IKKα and p100). Ectopically expressed NIK seems to spontaneously form oligomers in which these bindings of the N-terminal to the C-terminal regions in each NIK molecule are apparently disrupted, and display a high level of constitutive activity [Lin et al. 1999]. The binding of the NIK C-terminal region to TRAF2 (as well as to other TRAF's) most likely participates in the activation process of NIK. However, its exact mode of participation is unknown.
There is likewise rather limited information yet of the downstream mechanisms in NIK action. Evidence has been presented that NIK, through the binding of its C-terminal region to IKKα can activate the IκB kinase (IKK) complex. It has indeed been shown to be capable of phosphorylating serine-176 in the activation loop of IKKα, thereby activating IKKα [Ling et al. 1998]. Consistent with such mode of action, studies of the mechanisms accounting for the deficient activation of NF-κB by the LTβR in aly/aly mice murine embryonic fibroblasts (MEF's) indicated that NIK mutation ablates activation of the IKK signalosome and the consequent phosphorylation of IκB [Matsushima et al 2001]. These findings were not supported, however, by the analysis of MEF's derived from NIK −/− mice. Although the NIK deficient MEF's are unable to manifest NF-κB activation in response to LTβ, they do seem to respond normally to it in terms of IκB phosphorylation and degradation [Yin et al. 2001]. According to these findings, NIK may not participate at all in the activation of the IKK complex by the LTβR but is rather involved by an as yet unknown mechanism in controlling the transcriptional action of the NF-κB complex after its translocation to the nucleus. There are also still uncertainties as to the way by which NIK triggers p100 phosphorylation and processing. Its ability to bind p100 directly through its C-terminal region and phosphorylate it suggests that p100 serves as a direct NIK substrate [Xiao et al. 2000]. Nevertheless, a recent study has suggested that NIK mediates p100 phosphorylation in an indirect way, through phosphorylation and thus activation of IKKα that in turn phosphorylates p100 [Senftleben et al. 2001].
Yamamoto and Gaynor reviewed the role of NF-κB in pathogenesis of human disease (Yamamoto and Gaynor 2001). Activation of the NF-κB pathway is involved in the pathogenesis of chronic inflammatory disease, such as asthma, rheumatoid arthritis (see Tak and Firestein, this Perspective series, ref. Karin et al. 2000), and inflammatory bowel disease. In addition, altered NF-κB regulation may be involved in other diseases such as atherosclerosis (see Collins and Cybulsky, this series, ref. Leonard et al. 1995) and Alzheimer's disease (see Mattson and Camandola, this series, ref. Lin et al. 1999), in which the inflammatory response is at least partially involved. Finally, abnormalities in the NF-κB pathway are also frequently seen in a variety of human cancers.
Several lines of evidence suggest that NF-κB activation of cytokine genes is an important contributor to the pathogenesis of asthma, which is characterized by the infiltration of inflammatory cells and the deregulation of many cytokines and chemokines in the lung (Ling et al. 1998). Likewise, activation of the NF-κB pathway also likely plays a role in the pathogenesis of rheumatoid arthritis. Cytokines, such as TNF-α, that activate NF-κB are elevated in the synovial fluid of patients with rheumatoid arthritis and contribute to the chronic inflammatory changes and synovial hyperplasia seen in the joints of these patients (Malinin et al. 1997). The administration of antibodies directed against TNF-α or a truncated TNF-α receptor that binds to TNF-α can markedly improve the symptoms of patients with rheumatoid arthritis.
Increases in the production of proinflammatory cytokines by both lymphocytes and macrophages have also been implicated in the pathogenesis of inflammatory bowel diseases, including Crohn's disease and ulcerative colitis (Matsumoto et al. 1999). NF-κB activation is seen in mucosal biopsy specimens from patients with active Crohn's disease and ulcerative colitis. Treatment of patients with inflammatory bowel diseases with steroids decreases NF-κB activity in biopsy specimens and reduces clinical symptoms. These results suggest that stimulation of the NF-κB pathway may be involved in the enhanced inflammatory response associated with these diseases.
Atherosclerosis is triggered by numerous insults to the endothelium and smooth muscle of the damaged vessel wall (Matsushima et al. 2001). A large number of growth factors, cytokines, and chemokines released from endothelial cells, smooth muscle, macrophages, and lymphocytes are involved in this chronic inflammatory and fibroproliferative process (Matsushima et al. 2001). NF-κB regulation of genes involved in the inflammatory response and in the control of cellular proliferation likely plays an important role in the initiation and progression of atherosclerosis.
Finally, abnormalities in the regulation of the NF-κB pathway may be involved in the pathogenesis of Alzheimer's disease. For example, NF-κB immunoreactivity is found predominantly in and around early neuritic plaque types in Alzheimer's disease, whereas mature plaque types show vastly reduced NF-κB activity (Mercurio et al. 1999). Thus, NF-κB activation may be involved in the initiation of neuritic plaques and neuronal apoptosis during the early phases of Alzheimer's disease. These data suggest that activation of the NF-κB pathway may play a role in a number of diseases that have an inflammatory component involved in their pathogenesis.
In addition to a role in the pathogenesis of diseases characterized by increases in the host immune and inflammatory response, constitutive activation of the NF-κB pathway has also been implicated in the pathogenesis of some human cancers. Abnormalities in the regulation of the NF-κB pathway are frequently seen in a variety of human malignancies including leukemias, lymphomas, and solid tumors (Miyawaki et al. 1994). These abnormalities result in constitutively high levels of NF-κB in the nucleus of a variety of tumors including breast, ovarian, prostate, and colon cancers. The majority of these changes are likely due to alterations in regulatory proteins that activate signaling pathways that lead to activation of the NF-κB pathway. However, mutations that inactivate the IκB proteins in addition to amplification and rearrangements of genes encoding NF-κB family members can result in the enhanced nuclear levels of NF-κB seen in some tumors.
IL2 is a protein of 133 amino acids (15.4 kDa) with a slightly basic pI. It does not display sequence homology to any other factors. Murine and human IL2 display a homology of approximately 65 percent. IL2 is synthesized as a precursor protein of 153 amino acids with the first 20 aminoterminal amino acids functioning as a hydrophobic secretory signal sequence. The protein contains a single disulfide bond (positions Cys58/105) essential for biological activity.
Mouse and human IL2 both cause proliferation of T-cells of the homologous species at high efficiency. Human IL2 also stimulates proliferation of mouse T-cells at similar concentrations, whereas mouse IL2 stimulates human T-cells at a lower (sixfold to 170-fold) efficiency. The involvement of IL-2 in autoimmunity is controversial (reviewed by O'Shea et al. 2002) It is recognized that IL-2 administration is associated with a variety of autoimmune disorders such as immune thyroiditis, rheumatoid arthritis and other arthropathies. However IL-2 deficient mice produce multiple autoantibodies, including anti-DNA antibodies. About half die of autoimmune haemolytic anemia and the survivors develop inflammatory bowel disease. Importantly, the pathology is corrected by the addition of exogenous IL-2. This indicates a role of IL-2 in maintaining peripheral tolerance.
IL2 is a growth factor for all subpopulations of T-lymphocytes. The IL2R-alpha receptor subunit is expressed in adult T-cell leukemia (ATL). Since freshly isolated leukemic cells also secrete IL2 and respond to it, IL2 may function as an autocrine growth modulator for these cells capable of worsening ATL.
IL2 also promotes the proliferation of activated B-cells. Such activity requires the presence of additional factors, for example, IL-4. In vitro IL-2 also stimulates the growth of oligodendroglial cells.
Therefore, due to its effects on T-cells and B-cells IL-2 is a central regulator of immune responses. It also plays a role in anti-inflammatory reactions in hematopoiesis and in tumor surveillance. IL2 stimulates the synthesis of IFN-gamma in peripheral leukocytes and also induces the secretion IL-1 TNF-alpha and TNF-beta.
The biological activities of IL2 are mediated by a membrane receptor. Three different types of IL2 receptors are distinguished that are expressed differentially and independently. The high affinity IL2 receptor constitutes approximately 10 percent of all IL2 receptors expressed by cells. This receptor is a membrane receptor complex consisting of the two subunits IL2R-alpha and IL2R-beta as the ligand binding domains and a gamma chain as a signaling component. IL2R-beta is expressed constitutively on resting T-lymphocytes, NK-cells, and a number of other cell types while the expression of IL-2R-alpha is usually observed only after cell activation. IL-2-alpha is, however, synthesized constitutively by a number of tumor cells and by HTLV-1-infected cells.
IL2 receptor expression of monocytes is induced by IFNγ so that these cells become tumor-cytotoxic.
Murine and human gamma subunits of the receptor have approximately 70 percent sequence identity at the nucleotide and amino acid levels. This subunit is required for the generation of high and intermediate affinity IL2 receptors but does not bind IL2 by itself. These two receptor types consist of an alpha-beta-gamma heterotrimer and a beta-gamma heterodimer, respectively. The gene encoding the gamma subunit of the IL2 receptor maps to human chromosome Xq13, spans approximately 4.2 kb and contains eight exons. Relationships to markers in linkage studies suggest that this gene and SCIDX1, the gene for X-linked severe combined immunodeficiency, have the same location. Moreover, in each of 3 unrelated patients with X-linked SCID, a different mutation in the IL2R-gamma gene has been observed.
X-linked severe combined immunodeficiency (XSCID) is a rare and potentially fatal disease caused by mutations of IL2Rγ chain, the gene encoding the IL-2R γ chain, a component of multiple cytokine receptors that are essential for lymphocyte development and function (Noguchi et al. 1993). To date, over 100 different mutations of IL2RG resulting in XSCID have been published. Recent gene knock out studies indicate a pivotal role of this gene in lymphopoiesis [DiSanto et al 1995].
The IL-2Rγ chain is a subunit of the IL-2, IL-4, IL-7, IL-9, IL-13, IL-15 and IL-21 receptor complexes wherefore it now dubbed as the ‘common γ chain’ (cγc).
EP0578932 relates to the whole common gamma chain and especially to the extracellular N-terminal domain.
Consistent with the involvement of IL-2 in autoimmunity there exists a need for a modulator of IL-2 activity for preventing or alleviating said diseases.